Huntington's disease is a devastating neurodegenerative disorder caused by translation of an expanded trinucleotide repeat in the huntingtin (HTT) gene into a neurotoxic protein. There is no effective treatment or cure, although researchers are developing small interfering RNAs to target and degrade mutant mRNA. Because the normal HTT protein is essential for neuronal survival, therapies aim to spare wild-type mRNA. It may be possible to specifically target the mutant mRNA if mutant and wild-type alleles are processed differently after transcription. Research indicates mutant and wild-type mRNA exhibit different stability and localization, but the mechanism is unknown. One possibility is mutant mRNA is processed into a different isoforms with different localization and stability. Mutant and wild-type HTT mRNA are present as two 3'UTR isoforms. Indeed, preliminary data suggest mutant mRNA is more likely than wild-type mRNA to be present as the longer isoform. The proposed work aims to determine the mechanism and impact of differences in mutant and wild-type HTT 3'UTR isoforms. Because the mutation is proximal to gene regions involved in alternative polyadenylation (APA), the expansion may disrupt mutant HTT APA and cause the shift to the longer isoform. If so, other mRNAs and the other allele should be processed normally. To determine if the change in isoform length is HTT-specific, the length of HTT and other APA mRNAs will be assessed in heterozygous human samples by qPCR and deep sequencing. To verify the expansion is responsible, isoform lengths will be measured by qPCR from a transfected plasmid with or without the expansion. To assess whether the HTT isoform length changes during or after APA, the length of HTT isoforms from the plasmid will be assessed right after or much after labeling newly transcribed mRNAs with a modified nucleotide. These studies are the focus of Specific Aim 1. The change in mutant HTT isoform length likely impacts downstream processing because the 3'UTR contains many motifs directing mRNA stability and localization. To test this, the decay and localization of mutant and wild-type HTT isoforms will be assayed in normal and disease cells by transcriptional arrest and qPCR (decay) or fluorescent in-situ hybridization (localization). To verify differences are due to the isoform length, isoform localization and decay will be assayed in cell models transfected with a single HTT isoform. These studies are the focus of Specific Aim 2. Together, these studies will elucidate differences in mutant and wild-type mRNA APA, decay, and localization. Allele-specific small RNAs could then be designed to target mRNA regions or cellular compartments unique to the mutant isoform, improving treatment of this devastating disease.